Mithramycins as transplanted tumor inhibiting agents

ABSTRACT

THE MITHRAMYCIN A IS USEFUL IN INHIBITING THE GROWTH OF TESTICULAR TUMORS IN MAN. THE MITHRAMYCIN A, B AND C ARE USEFUL IN INHIBITING THE GROWTH OF TRANSPLANTED TUMORS IN LOWER ANIMALS.

Feb. 29, 1972 B, A, SQBlN ETAL 3,646,194

MITHRAMYCINS AS TRANSPLANTED TUMOR INHIBITNG AGENTS Filed Aug. i9, 1968 4 Sheets-Sheet l PERCE N T TRANSM'ITTNCE Q INFQARED BSOEPTIUN SPECTRUM OF MTTHg''.

PERCENT TRANSMITTANCE Feb. 29, 1972 B. A SOB|N EVAL 3,646,194

MITHRAMYCINS AS TRANSPLANTED TUMOR INHIBITTNG AGENTS Filed Aug. L9, 1968 4 Sheets-Sheet i;

Q PERCENT TRANSM'TTANCE es Q WAVE NUMBERS 1N cM-i 1600 i400 |500 i200 H00 7 9 WAVE LENGTH 1N MIcRoNs N 0% S Q PERCENT TEANMmANcE Feb. 29, 1972 B, A 505m; ETA'. 3,646,194

MLTHRAMYCINS AS TRANSPLANTED TUMOR INHIBITING AGENTS Filed Aug. 19, 1968 4 Sheets-Sheet 7 PERCENT TRANSMITTANCE 9 l0 WAVE LENGTH IN MICRONS F 5 INFRARED ABSQRPTIUN pEcTRUM 0F MITHQAMYCIN-MANHYDRoU) WAVE NUMBERS IN @wi-1 i500 Mw |300 i200 PERCENT TRANSMITTANCE Feb. 29, 1972 BA, 503m ETAL 3,646,194

MI'IHRAMYCINS AS TRANSPLANTED TUMOR INHIBITING AGENTS Filed Aug. L9, 1968 4 Sheets-Sheet L' PE RCEINT 'IPANSMITTANCE PERCENT TRANSMIT'TANCE United States Patent lce 46,194

Patented Feb. 29, 1972 cludes within its scope these products in dilute forms, as MI MYCINS AISPLANTED TUMOR crude concentrates and also the pure crystalline forms th f. Th .t IHRA INHIBITING AGENTS creo e novel mi hrairiycin A is useful in inhibiting the growth of malignant testicular tumors in man. Mith- Ben A' Sobm Manhasset NY" Joh. B Routlen Ifyine 5 ramycin B and mithramycin C are useful in inhibiting the Conn., Koppaka V. Rao, Cambridge, Mass., William s Marsh wanaque NJ and Aline L Garretson East growth of transplanted malignant tumors in lower ani- Lansing, Mich., assigmors to Pfizer Inc., New York, mals' Streptomyces plzcatus (Parke-Davis & Company, Cul- Continuation-impart of application Ser. No. 617,442, Dec. ture B'llrall, DetrOit, Mich NO- 04918) iS known 'lo Pro 19, 1966, which is a continuation-impart of application duce three weakly basic antibiotics, amicetin, plicacetin Sel'- No- 317,399, C- 15, 1963, Which ill tllrll 1S a and bamicetin (Haskell et al., J. Am. Chem. Soc. 80, 743- comm' ation-impart 0f applications Ser- N0- 696,676, 747 (i958) and British Patent 707 332) The products Nov. 1957 and Ser. No. 39 572 June 29 1960. This applicahzon Ag. 19 1968, SerINOL/,SSSM of the present invention, on the other hand, are acidic Int C] Alk 21/00 substances, the properties of which are described below. U.S. Cl. 424-119 7 Claims 15 SUMMARY OF THE INVENTION Mixtures of mithramycins A, -B and C are produced BSTRACT 0F THE DIsCIOSURE during the cultivation, under controlled conditions, of The'mlhfamyoin A S Useful in lnhlbitllg the growth 20 either a new species of microorganism which has been of teslculal' tumors in m2111- The mithfamyoin A, B and designated Streptomyces argllaceus or a new strain of a C are useful in inhibiting the growth Of raIlSplaIled species known as Streptomyces plcatus. Mixtures of these tumors in lower animals. closely related antibiotics appear to be always coproduced in the fermentations described herein. (Mithramycin A 25 is the major component). The mixture (referred to as the `CROSS-REFERENCE TO RELATED mithramyciii complex in our copending application Ser. APPLICATIONS No. 39,572) produced by the new strain of S. plicatus iS This application is a continuation-in-part of copending made .p of abqut 85% 98% mlthramycmA and 15.% application Ser. No. 617,442 led Dec. 19, 1966, which is, 2% mlthramycms B and/o1' C. The se microorganisms in tum a contnuatomimpart of application ser. No. were isolated from soil samples and identified by plant- 317,399 tiled Oct. 15, 1963, which is in turn a continuationmg and te,stmg.cu1tl,lres theeof on mefla non nauy used iii-pari of appiieaiion ser. No. 39,572 nied June 29, i960 for Suc@ llenflatlon Whlle C9H1Paf-mg the Cultural and application Ser. No. 696,676 iiled Nov. l5, 1957, all characteristics with those described. m Wilkiman and of which are now abandoned* Lechevalier, Actinomycetes and Their Antibiotics, 1953. A culture of the new species, Streptomyces argllaceus, has

BACKGROUND OF THE INVENTION been deposited with the American Type Culture Collection,

This invention relates to the new and useful fermenta- Washington, DC., under the number, ATCC 12956, while tion products Called mithramycin As mithramycn B and a culture of the new strain of Strepomyces plzcatus has mithramycin C, to the salts thereof and to mixtures of been deposited with the same culture collection under the Said products and their salts, to their production by fernumber ATCC 12957. It is identied in the culture colmentation, to methods for their recovery and concentraleotlon of Chas- PZeI & Co, IUC- as Isolate 415A-11028- tion from crude Solutions, such as fermentation broths, `The cultural characteristics of the new species, Streptoand to processes for their purification. The invention inmyces argillaceus, are set forth in the following table,

TABLE I [Streptomyces Argillaceus sp. nov. ATCC Number 12956] Aerial Myeelium and Medium Amount of Growth Sporulation Soluble Pigment Remarks Pridhams yeast- Chains of spores straight or wavy or spirals at dextrose agar. tip, many chains in clusters; spores almost spherical to oval to cylindrical averaging 1.6-2.01 x 1.0-1.3, but varying from 1.3 x 1.3; to 2.0 x 1.0i. Glucosc-Asparaginc Poor to poor or moderate.. Almost none; gray-white None Vegative mycelium yellowish tan to brown; agar. to dark olive. nrrginal area meruloid; reverse yellowish tan o rown.

Skimmed milk do Sparse; yellowish white... Drab Vegetative mycelium yellowish tan to dark brown; milk coagulated, no peptoriization in one tube, two other tubes had rio coagulation or hydrolysis; pH change from 6.4 to 5.7.

Glucose agar Moderate Sparse to moderate or Dark blaekish brown Vegetative mycelium yelloWish-grayish tan;

moderate on lower reverse yellowish tan to brownish black. portion of slant; pale gray. Nutrient agar Poor Rare; grey whate Yellowish brown Vegetative mycelium colorless; reverse colorless. Synthetic agar Moderate Sparse to moderate; None Do.

grayish-white.

Calcium malate do Moderate; pale gray Slight yellowish Vegetative mycelium colorless where visible;

becoming darker'. soluble pigment. reverse grayish tan; malate digested.

Cellulose do Good; brownish gray. None Potato slants Moderate to good Very sparse to moderate Dark olive brown Vegetative mycelium dark olive-black; reverse (on one tube). dark olive-brown. Starch plates Sparse Good; grayish white to None Vegetative mycelium colorless where visible; dark gray. reverse creamy White to yellowish gray; partial htyd rolysis (zone of hydrolysis 2.5 cm. in diame er Gelatin plates Moderate Good; gray to grayisli- Dark Vegetative mycelium not visible; colonies dewhite. pressed, marginal area smooth, lobed, more or less coneentiically zoned; reverse dark blackish brown; no liquefaction.

Dextrose-nitrate Sparse Sparse; white Very pale yellow Vegetative mycelium creamy white; no reduction broth. of nitrates to nitrites.

Einersons agar Moderate Sparse to moderate; Dark brown Vegetative mycelium colorless where visible.

white to pale gray.

wherein the description given is based upon readings made after two Weeks of growth. Classiiication of this new species was made by Dr. .lohn `B. Routien, who proposed the name Streptomyces argillaceus. The culture ATCC agar (Int. Jr. Sys. Bact.), glycerol-asparagine agar (Int. Ir. Sys. Bact.), peptone-yeast extract iron agar (Int. Jr. Sys. Bact.), tyrosine agar (Int. Jr. Sys. Bact.), carbohydrate utilization (Int. Jr. Sys. Bact.), gelatin (Gordon 12956 has been designated the type culture of this new 5 &Mihm, Jr. Bact. 73: 15-27, 1957), skimmed milk (Difco species. product), starch agar (20 gm. starch, 0.5 gm. NH4Cl, The cultural characteristics ofthe mithramycin-producagar gm., pH 7.0, distilled H2O to 1 liter), Waks- :lng culture Isolate BA-1l028, previously identified as a mans starch agar A (Waksman, The Actinomycetes, new strain of Streptomyces tanashiensz's, are described lin 1950), basal medium plus various S sources (Kuster & Tlable II which presents a s ide-by-side comparison iis/1th l0 Williams Applied Microbiology 12, 46 52y 1964) dex (gle'naws Strain 04918) and S' mms 'ens'sf trose nitrate broth (Waksman, The Actinomycetes, i950),

organic nitrate broth (Gordon & Mihm, Jr. Bact.),

Culture BA-ll028 was compared with S. plrcatus C k, W k Th A t. t 1950 1 (Parke-Davis strain 04918) described in British Patent Zape a smanak e C mtmefs gucose' 707,332, published Apr. 14, i954, and S. ranashiensfs 15 aspafagme agar (W Smal" T clmomyces 1950) (NRRL B-2122). The media or tests used were as follows: nument agar (Waksman The Actmolnycetes 1950) and tryptone-yeast extract broth (Shirling & Gottlieb, Inter- Pola-0 Plugs (Waksrnan, n The Actinomycetes, 1950). national Ir. Systematic Bacteriology 16 (3): 3l3-340, Methods Were hOSG given the Int. lr. SYS- BaC- Paper 1966), yeast extract-malt extract agar (Int. Jr. Sys. Baer), of 1966- Temperature 0f mcubatlon was 28 C. The oatmeal agar (Int. Jr. Sys. Bact.), inorganic salts-starch 20 results are assembled in Table 1I.

TABLE II [Streptomys plcatus, ATCC No. 12957] Medium S. tanashr'eusis S. plcatus 13A-11028 Tyrosine agar Meianin No melanin; pink soluble pigment afteretdays No melanin.

growth on medium. Tryptone yeast extract broth- Melanin No melanin Do. Dextrose nitrate broth Nitrates produced Nitrates produced. Nitrates produced. Organic nitrate broth. do do Do. Skimmed milk Coagulation, pcptouization; tan soluble Coagulation slow and partial, some peptini Coagulation slow and pigment. zation, slight pink to tan soluble pigment. partial, some peptonization, pink to tan soluble pigment.

Gelatin Good liquel'action Good liquefaction Good lqucfaction.

HZS production (lead acetate Strong from cysteine, NanSzO and trypto Strongin cysteine,proteosepoptone,NazSZOg,

Same as S. pZt'catu-s strips used for detection) phane and from peptone-iron agar plus peptone and peptone-iron agar plus yeast except no B2S from yeast extract. extract. NagSOz. Starch agar No hydrolysis Very narrow zone Narrow zone. Waksmans starch agar Strong hydrolysis do Strong hydrolysis. Carbon utilization:

Glucose Yes Yes Yes. Arabinose 2+ Dulcitol l. l. 2+. l l.

2+. -l. Wakmans starch agar:

Aerial mycellum-- Gray Gia Gray. Reverse Dark gray Ycllowisli-brown. Soluble pigmen Light brown Lacking. Inorganic salts-starci Aerial mycelium Gray. Reverse Grayisli-olive. Soluble pigment Lackin Lacking Lacking. Yeast extract-malt extract-z Aerial mycelium Gray Gray Gray. Reverse Light brown. Light brown Dark gray. Soluble pigment Dark bonvn L Lacking. Synthetic:

Aerial mycelinm Lacking Seant; gray Scant; gray. Reverse Colorless White White Soluble pigment Lacking Lacking Lacking. Nutrient agar: Aerial mycelium Gray White White. Reverse Pale tan Cream Cream t0 brown. Soluble pigment Light brown Lacking Lacking. Glucose asparagine agar:

Aerial mycelium Gray Scant; gray Lacking. Reverse Brown- White Ye.loW. Soluble pigment Light brown Lacking Lacking. Oatmeal agar:

Aerial rnylium Gray Gray Gray. Reverse Brown Yellow. Soluble pigment Gray-brown Lacking Lacking. Iotatoe slices:

Aerial myceliurn Lacking Gray Gray. Reverse dark brown t0 black Yellowish to yellowish-gray Yellowish to yellowish-gray. Soluble pigment Black very pale gra Very pale gray.

Microscopio features; inorganic saltsfstarch. mostly Chains Sti-% RA,

25-50% spira: itl-50 spores per chain.

RA type, 10% spira; 1li-5o spores per chain.

Yeast extract-malt extract. Chains mostly flcxuous, some RA type. Chains mostly RA, some spira Chains mostly RA we Oatmeal agar Spores smooth by electron microscopy Spor-es smooth by electron microscopy Spores spiny by elecirou microscopy.

S. plcatus was quite different from S. tanaslzz'enss in the following ways: producing no melanin; differences in production of H28; seven differences in carbohydrate utilization; differences in reverse and soluble pigment on potato and several other media; difference in shape of chains of spores.

BA-11028 was more similar to S. plicatus than to S. tanashiensis, though it showed the following distinctions from the former: no H28 produced from Na282O3; strong hydrolysis of starch; a few differences in carbohydrate utilization; slight differences in color of reverse and of soluble pigment on some media; spiny spores. The slight difference in production of H28 and of degree of starch hydrolysis seem minor, and the only absolute difference in carbohydrate utilization is on rainose. The greatest difference is in the surface markings of the spores, and several papers in the literature indicate that the type of marking may not be absolute for some species.

Because of these facts and the fact that S. plz'catus and BA-ll028 are so similar, it is concluded that BA-11028 is a strain of S. plcatus.

It is to be understood that the present invention is not limited to use of the aforesaid organisms or to organisms fully answering the above descriptions, which are given only for illustrative purposes. It is especially desired and intended to include the use of naturally occurring or artiicially induced mutants and/or variants, such as those which can be produced from the described organisms by various means, including x-radiation, ultra-violet radiation, treatment with nitrogen mustards, and the like. For example, a subculture of BA-11028A which has been found to produce mithramycin is described below. It is identified in the culture collcetion of Chas. Pzer & Co., Inc. as Isolate BA-ll028A.

BA-11028A is a culture derived from BA-ll028 by plating out single spores of the latter and selecting different types of colonies. One of those selected was designated BA-llOZSA. Since it produced mithramycin, the same compound produced by 13A-11028, it was concluded that it was derived from that strain and was not a contaminant.

It was also grown on the media described in Table II at the same time as were the other three cultures. Though it did not look exactly like BA-11028, it is concluded that it still should be designated as a variant of S. plcatus.

The differences between BA-l1028 and BA-llOZSA are given as follows: very weak reduction of nitrate to nitrite by the latter; H28 produced from Na2S2O3 (as by S. plcatus) by the latter; no growth on rhamnose. On rafinose and sucrose the growth was i for BA-11028A compared with-for BA-1l028, while on mannitol and sorbitol it was-for BA-l1028A and i for BA-1l028. Additionally, BA-11028 showed more brown color in the reverse on several media than did BA-11028A.

We wish also to include any organism, regardless of its appearance or physiological behavior, that may be developed by means of transformation, transduction, genetic recombination or some other genetical procedure, using a nucleic acid or an equivalent material from the herein described species, whereby it has acquired the ability to produce the elaboration product here described or to carry on the biochemical change here described.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings illustrate characteristic in frared absorption spectra of the various products of this invention. FIGS. l, 2, 3 and 4 show the spectra of mithramycin A, mithramycin B, mithramycin A (anhydrous) and mithramycin C, respectively.

DETAILED DESCRIPTION OF THE INVENTION This invention includes within its scope processes for growing the microorganisms S. argllaceus ATCC 12956 and S. plicazus ATCC No. 12957. The cultivation of these microorganisms preferably takes place in aqueous nutrient media at a temperature of from about 24-30 C., and

under submerged, aerobic conditions with agitation. Nutrient media which are useful for such purposes include a carbohydrate, such as sugars, starch, glycerol, corn meal; a source of organic nitrogen, such as casein, soy bean meal, peanut meal, wheat gluten, cotton seed meal, lactalbumen, enzymatic digest of casein. A source of growth substances, such as distillers solubles, yeast extract, molasses extract residues, as well as mineral salts, such as sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, and trace minerals such as copper, zinc and iron, may also be utilized with advantageous results. A particularly useful and preferred medium is one containing dextrose, soybean meal and potassium phosphate. If excessive foaming is encountered during fermentation, anti-foam agents, such as vegetable oils, may be added to the fermentation medium. The pH of the fermentation tends to remain rather constant, but if variations are encountered, a buffering agent such as calcium carbonate may also be added to the medium.

Inoculum for the preparation of the mithramycin products may be obtained by employing growth from slants of the aforesaid microorganisms on such media as Emersons agar or beef lactose. The growth may be used to inoculate either shake flasks or inoculum tanks, or alternatively, the inoculum tanks may be seeded from the shake asks. The growth of the microorganism usually reaches its maximum in about two or three days. However, variations in the equipment used, aeration, rate of stirring, and so forth, may affect the speed with which the maximum activity is reached. In general, the fermentation is continued until substantial antimicrobial activity is imparted to the medium, a period of from about 24 hours to about 4 days being sufficient for most purposes. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about 1A. to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained throughout the transfer of the microorganisms and throughout their growth.

After growth of the microorganism, the mycelium may be removed from the fermentation broth by filtration, centrifugation, or the like. Thereafter, the mithramycin products may be recovered by several different procedures. For example, the filtered broth may be used as is or it may be dried. Preferably, however, the products are further puried. For example, they may be extracted from aqueous solution at substantially neutral to acidic pH conditions, i.e. pH of about 2 to 7, by means of a variety of water-immiscible organic solvents, including the lower alcohols, aromatic alcohols, esters, ketones, halogenated hydrocarbons and mixtures thereof. Examples of these are n-butanol, sec-butanol, amyl alcohol, hexyl alcohol, benzyl alcohol, ethyl acetate, methyl isobutyl ketone, chloroform, ethylene dichloride and trichloroethylene. Of course, the solubility in these solvents varies somewhat, the lower alcohols and methyl isobutyl ketone being preferred for best results. The mithramycin products may be extracted from most solvent solutions back into water by concentrating the solvent extract and adding a non-solvent for the antibiotic, such as petroleum ether, cyclohexane, and the like. By adjustment of the pH to the range set forth above, the product may be reextracted into one of the water-immiscible solvent indicated above. Upon drying the solvent and concentrating the solution, the product separates in crude form.

A more puried form of the mithramycin products is obtained by chromatographing a concentrate resulting from solvent extraction with one of the solvents indicated above, such as methyl isobutyl ketone or butanol. The extract is preferably chromatographed on Florisil (an activated magnesium silicate available from the Floridin Co.) or on an acid-washed alumina column which is then developed with suitable solvents to isolate the mithramycin products. The following flow sheet shows a suitable method of recovering the mithramycin products from the fermentation broth produced by each of the microorganisms previously described.

S. a rgilla cous S'. plicatus Fermentation.

Filter broth.

Adjust filtrate to pII (-7).

Solvent extraction (butanol).

Fermentation Filter broth Adjust fiiltrate to acid pH Solvent extraction (methyl isobutyl ketone) Concentrate extract. Concentrate extract Wash with isopropyl other.

Filter Alumina column.

Develop with CHClf-MeOH Evaporate to dryness Add water and dry Fraction I Aeidiil'y filtrate Solvent extraction (ethyl acetate) Mithramycin products When the mithramycin mixture is produced with S. argllaceus, the alumina column, with the product adsorbed thereon, may be developed with a chloroformmethanol system. Upon evaporation of the eluate, addition of |water and drying, a mixture of pure microcrystalline mithramycins is obtained.

On the other hand, with S. plicafus it has been found that certain contaminating products of relatively low biological activity are coproduced, making the recovery procedure somewhat more complicated. These products are herein designated as fraction I and fraction II. In this case, fraction I may be recovered by concentrating the extract resulting from solvent extraction of the filtered fermentation broth. Upon acidification of the filtrate and development thereof on an acid-'washed alumina column, fraction II is obtained in the eiiiuent. Elution of the alumina column with a suitable solvent, such as methanol-ethyl acetate, results in a mixture of pure microcrystalline mithramycins A, B and C.

The substance referred to in our first tiled application, Ser. No. 696,767, as PA 144 is identical with the substance referred to in Ser. No. 39,572 as mithramycin complex and referred to in Ser. No. 317,399 as a mixture of mithramycins A and B. In this mixture the proportion attributable to B is now sometimes found to consist of mithramycin B and/ or mithramycin C. Mithrarnycin B was not described in our rst filed application. It is produced in relatively minor amounts compared to mithramycin A and mithramycin C and in some instances may not be produced at all or only in very small amounts. It possesses substantially the same biological activity as mithramycin A, the predominant component, and the mithramycin mixtures produced herein. It appears to be chemically of related structure to mithramycin A in view of the similarity in physical properties of the two substances. Mithramycin C was not described in our earlier led applications. It possesses less biological activity than does mithramycin A or mithramycin B although it appears to structurally relate to both these materials. Its behavior on paper chromatography is very similar to that of mithramycin B, so similar in fact that the two cannot be distinguished by this method. However, despite this, both mithramycin B and mithramycin C are distinct substances and are different from the materials t0 which reference was made in our earlier application Ser. No. 696,676 as Fractions A and B and referred to in Ser. No. 39,572 as Fractions I and II. The latter substances have only a fraction of the biological activity of the mithramycin mixture.

A recovery procedure particularly `adapted to recovery of the mithramycins from fermentation broths is represented schematically in the following ow sheet. The method involves adsorption of the active constituents on activated magnesium silicate or fullers earth and elution therefrom. When employing fullers earth as absorbent, acetone or ethyl acetate may also serve for elution.

W hole- Broth l 1- pI- 4.0 and Filter Solids Magnesium Silicate Colunin Wash with Methanol Eluto with Methanol Effluent Discarded Solids Ldw`Concentrate Discarded Extract at pH 4.0 with Ethyl acetate Ratnate Ethyl acetate Concentrate Discarded to Purification The ethyl acetate concentrate containing the mixture of mithramycins can then be further puried, if desired or necessary, by column chromatography or by countercurrent distribution employing the solvent system 3% phosphate buffer pH 7.0-7.5 ethyl acetate in an apparatus providing for 200 transfers. The active material concentrates in tubes -160 and is recovered therefrom by concentration of the combined ethyl acetate phase, treatment of the concentrate with ether and extraction with water. The aqueous extract is then frozen and dried from the frozen state.

Pure crystalline mithramycin A, mithramycin B and mithramycin C can be separated from the crude ethyl acetate concentrate from the magnesium silicate or fullers earth recovery procedure by chromatography employing a silicic acid or a silicic acid-cellulose powder column. Elution of this column rst with ethyl acetate followed by ethyl acetate containing 5% by volume of methanol provides in the first fraction, virtually with the solvent front, mithrarnycin A. Mithramycins B and C are held more tenaciously by the adsorbent and are eluted only when the methanol-ethyl acetate solvent mixture is employed. The products are recovered from the appropriate eluate fractions by concentration and crystallization from butyl acetate, amyl acetate or acetone.

The mithramycin A thus obtained is a dihydrate. It is converted to the anhydrous form by drying at C. for 24 hours over phosphorous pentoxide under reduced pressure, eg. at 0.05 mm. of mercury.

The molecular formula of mithramycin A calculated from the following analytical data on the anhydrous form is C52H1s024 Hydrolysis (0.1 N HC1) of mithramycin A shows it to be composed of an aglycone (C21H24O9), three sugars (A:C7H1404; B=C6H1204 and C=C6H1204) Table III which appears below contains a tabulation of the physical properties of mithramycin A, hydrated and anhydrous forms, mithramycin A sodium salt and mithramycins B and C. Table IV contains a listing of color reactions observed with mithramycin A. Significant differences in the infrared absorption of mithramycins A and B occur in the region 1228 to 1720 cml.

n Mithraniycin A Mithl'ainycin A Property M1thramyein A sodium salt (anhydrous forni) Mitlu'ainycin B Mitliramycin C Appearance Yellow crystalline solid. Bright yellow long Yellow crystalline solid Orange yellow micro- Bright yellow crystalrectangular psisms. crystalline solid. line solid. Melting point C.) 1851-187 270-275 decomposed.. 184187 170-75o decomposed.-. 18284. Behavior under ultra- Dull yellow Bright yellow Brilliant yellow violet light. uorescenee. iiuorescence. fluorescence. Ultra-violet spectrum: 230 mu EL@ =220110 240 mu Eli@ =183 230 mu EQ =220+10 230 mu Ei =190 230 mu El@ =220|01 lm. 1Wm. 1 cm. 1 cm. 1 cm. (methanol) 278 mu E1 um. =480+20 285 mu hgh =475... 218 u1u =500+20. 290 mu =312 280 mu 1,3%* :550-1-25 Ultra-1110100 spectrum; 240 mu E =1s5 230 mu 1:. 177 230 mu =210 L7. m. 1% 1% (Water) 285 rnu EIT;- -289 280 mu ,1i-m 286 280 mu Em' -294 cui. A 1 cm. 41115111015 c3, 55.1%, 7.2, o), (bio, 54.e5;1"i, 6.05.-- (o3 11 5(77011 r, 7.2; o, C, 55.22; H, 7.13; o, o, 55.25; 11,099; o,

7. y 1 erence y erence 37.65 b difference 37.76 Equivalent weight 1115115 (by dlerenm' Specific rotation [alDg 58 +38.8 I in mefihanol (05012127 36 63 S03 s47 901 950 06 0 nlirare spectra 7 ,7 8 ,852, 9 8, 952, 983, 722, 743, 808, 847, 903 848 905 980 10 20 1 my), J 018, 1,000,1,004,1,110, 10021071.1120. 048. 210001003, 1107011.106/1 233 1, 100, 1, 22s, 1, 259, 1, 170, 1, 236, 1, 266, 1, 116, 1, 160, 1, 22s, 1, 266, 1, 299, 1 40g' 1, 203, 1, 320, 1, 310, 1, 300, 1, 330, 1, 374, 1, 260, 1, 322, 1, 347, 1, 515, 1, 634, 1 705' 1,396,1,1101F 1, 515, 1,631, 1, 724, 1, 36s, 1, 430, 1, 440, 3, 436, 0m -1 (see' 1, 550, 1, 626, 1, 720, 2, 890, 3, 413 0m -1 1, 500, 1, 585, 1, 63o, Figure 4) 2, 850, 2, 910, 2, 050, (S00 1gure 3) 1, 720, 2, 850, 2, 90o, 3,890 c .-1 (See 2, 940, 3, 380 om .1 Figure (see Figure 2) E Calculated for CHOZHC, 55.70; H, 7.19,() (by difference), 37.10 CgHysOgl: C, 57.55; H, 7.08; 0 (by difference), 35.37.

TABLE IV.-COLOR REACTIONS OF MITHRAMYCIN A Property: Mithramycln Alcoholic ferric chlo ride Green. Aq. NaOH Bright yellow. Conc. H2804 Deep brownish violet. Aq. Na2S2O4 No change in the yellow color. 2,4-dinitrophenylhydrazine Brick red precipitate.

Diazonium reagent Couples to form a red color.

The mithramycins are acidic substances and readily form salts with bases, c g. alkali and alkaline earth metal hydroxides and carbonates. They also form complexes with polyvalent metal ions and are, therefore, useful for inactivating metal ions, e.g. in biological experimentation and in metal separations.

Mild hydrolysis of mithramycin A with 2%-10% acetic acid at 100 C. for 20-30 minutes 0r with 0.1 N HC1 at room temperature overnight produces a product which appears to be identical to mithramycin C.

Mithramycins A, B and C may be assayed individually or as mixtures by observing their activity against various tumors, such as Sarecoma 180 in mica and H.S. No. l or C.A. 755 in rats, in tests more fully described hereinafter. However, a more convenient assay involves the use of the microorganism B. subtilis as the test organism. A serial dilution type of assay is employed, with the last dilution at which a distinct zone of inhibition occurs, being the number of dilution units/ ml. of sample. By comparing this with the result obtained in the same test with a puriiied standard, the potency of the sample is readily computed. In the assay of mixtures, the C component, because of its lower biological activity relative to that of mithramycins A and B, contributes relatively little to the observed activity. The principal contribution arises from mithramycin A, the predominant component. The remainder of the observed activity is due to the B and/or C components which may be present. The contribution of B when B is present usually outweighs that from mithramycin C.

A more convenient and reliable assay method comprises thin layer chromatography on silica gel with the system m e t h yl ethyl ketone:methanolzisopropanol (821:1). In such an assay of a mixture of mithramycins A, B and/or C, the separated mithramycin B and/or C d Calculated for C52H75O24Na2H2O C, 54.16; H, 6.60; cCalculated for spot is removed from the plate, eluted with methanol and the optical density of the eluate read at 280 m11. Assay of four mixtures of mithramycins A, B and/or C isolated from S. plicatus fermentations by the procedure of Example VII gave the following values for mithramycins B and/or C: 2.0, 2.8, 3.8 and 4.8%. These values are representative for the isolated naturally produced mixtures.

The mithramycin mixture and the iiltered broth resulting from cultivation of the above-described microorganisms were tested to determine their activity in inhibiting the growth of transplanted tumors in mice and rats. In one such test, Swiss mice of the same sex, weighing 1-8 to 22 grams, were employed. These mice were implanted with a Sarcoma tumor originating at the Southern Research Institute, Birmingham, Ala. Tumor fragments of from one to two mm. dimensions wereV cut from nonnecrotic portions of the donor tumor and implanted into the axillary region of each test mouse. Injections of the mithramycin mixture were begun 24 hours after tumor implantation and continued two times daily for 7 days. The animals were weighed on the rst, fourth and eighth days to provide a lmeasure of the toxic effects of the drug. Twenty-four hours after the last injection, the animals were sacrificed and the tumors excised and weighed. The results of this test employing ltered fermentation broth, obtained as in Example II, administered twice daily in 0.5 ml. doses as a source of mithramycin products are presented in Table V.

In like marmer, the activity of a purified mixture of microcrystalline mithramycins was tested against Sarcoma 180 in mice. The results of these tests are set forth in the following table:

TABLE VI [Sarcoma 180 inhibition] Mortality Percent Dose, mgJkg. rate inhibition 1.0 0/7 49 Controls 0 The anti-tumor activity of the mithramycins was further evaluated against human tumors in heterologous hosts. The human Sarcoma H. S. No. 1, which had been cultivated Yby Chens technique (Cancer Research, 14, 660 (1954)) was used. yRats bearing tumors were sacrificed, the tumors removed and cleansed in a solution containing penicillin and dihydrostreptomycin. The tumors were then minced and suspended in buffered glucose Ringer solution at a concentration of 80% tumor tissue. Rats which had been previously exposed to 150 R of X-ray radiation and treated with cortisone acetate were then injected subcutaneously in the flank with one rnl. of the above suspension. Animals implanted with the suspension of HES. No. l received their first treatment with mithramycin complex by intraperitoneal injection 24 hours after implantation, and daily doses thereafter only 24 hours prior to sacrilice on the 13th day. In every case, control groups of rats were maintained. Tumors of reproducible size within the usual biological limits were obtained in the control animals. At the time of sacrifice, the tumors were excised, divester of connective tissue, and weighed. The results of these tests, using ltered fermentation broth containing mithramycins A, B and C are set forth in the following table:

TABLE VII [H.S. No. 1 inhibition] Mithramycin Mortality Percent broth filtrate rate inhibition In like manner, the pure crystalline mithramycin mixture is tested, and the results obtained tabulated as in A similar te'st conducted with a different tumor, HEP No. 3, also demonstrated the tumor-inhibiting properties of the puriled microcrystalline mithramycin mixture as illustrated by the following:

TABLE IX [HEP No. 3 inhibition] Mortality Percent Dose, ing/kg. rate inhibition Mithramycins A and B exhibit signiiicant activity against the mammary adenocarcinoma CA-755 when tested according to the procedure of Celhorn et all, Cancer Research, Supplement Ill, page 38 (1955). These results appear in Tables X and X11-A.

TABLE X (CA 755 inhibition] Mithramycin broth Mortality Percent filtrate dilution rate inhibition 1-4 l/ l0 7G Controls 0/10 0 That mithramycins A and B are both active antitumor agents and contribute about equally to the observed activity whereas mithramycn C is much less active and requires much higher doses is apparent from the following data. The test described above (Table X) was conducted employing the pure crystalline forms of mithramycins A, B and C. These results are arranged in Table XI-A.

TABLE Xl-A [CA .55 Inhibition] Dose, Number of Percent Compound nig/kg sui-vivais inhibition Mithramyein A 0. 5 8/1() 70 Mithramycin B 0. 9,/10 46 D0 .i 0. '75 lOl/l0 5l.

Further and significant antitumor activity of the A and B components has been demonstrated against H.Ep. #3 (a human epidermoid carcinoma) tumors in the legs of mice.

TABLE XI-B [H.Ep. #3 Inhibition] Dose, Number of Percent; Compound ing/kg. survivals inhibition Mithramycin A-.. Y 1. 25 5/8 42 Do 1,00 6/8 74 0. 7'5 5/8 47 0, 50 8/8 62 1. 50 5/8 45 1.50 8/8 S8 l 00 8/'8 42 0. 75 8/8 59 In the treatment of H.Ep. #3 tumor in new-born Swiss mice, Merker, et al. (Antimicrobial Agents and Chemotherapy, pages 148-158, 1961) found a mixture of mithrarnycins (approximately A-10% B and/or C) to be the most effective agent for inhibiting the tumors growth.

When filtered fermentation broth containing the crude mithramycins was tested against 1.*1210, a human leukemia in mice, the following results were obtained.

TABLE XII.-Ll 2 l 0 IN HIBITION xFiltered broth-undiluted Life span-'69.8% `of controls The relatively high activity obtained with the mithramycins from iiltered fermentation broth suggests the possibility of synergism between mithramycins A and B and the products produced therewith, i.e. fractions I or II.

The mithramycins are relatively toxic substances. A lethal dose in mice was found to be of the order of 2000 mcg/kg. of body weight. The substance was less toxic by the subcutaneous route. In seven-day chronic toxicity tests on healthy mice to which a puried microcrystalline mithramycin mixture (about L75-5 IB and/ or C) was administered according to the regimen employed in the Sarcoma A test (two 0.5 ml. injections daily of aqueous solutions containing appropriate drug concentrations), the LD50 was found to be 1500 mcg./ kg.

vDespite their relatively high toxicity, mithramycins A and B and mixtures thereof have a very favorable therapeutic index, since doses of 15 4to 250 mcg/kg. of body weight are highly effective in inhibiting transplanted tumors in animals. As regards humans, doses equivalent to about 25 mcg/kg. of body weight/day are recommended dosage for mithramycin A. This is accomplished by administering 25 mcg./kg./day or 50 mcg/kg. every other day.

Although mithramycin mixtures and the A, B and C components thereof may be administered parenterally, either as an aqueous solution or dissolved in physiological saline, various other types of pharmaceutical preparations may advantageously be compounded therewith. These preparations may include Pboth liquid diluents suitable for extemporaneous preparation of solutions prior to administration. Illustrative of such diluents are: propylent glycol, diethylcarbonate, glycerol, sorbitol, etc. While other routes of administration are possi-ble, the parenteral routes are generally preferred, The various dosage forms 13 may contain buffering agents, as well as local anesthetics and inorganic salts to afford desirable pharmacological properties.

The techniques of isolated perfusion and regional perfusion, particularly the latter, have shown promise when preparations of mithramycin products are used as the chemotherapeutic agent.

For most purposes, the solid preparations of mithramycin products, i.e. of mithramycine A, mithramycin B and mixtures thereof, should contain the compounds in an amount of at least 0.05 mcg/mg. of the composition. Liquid preparations containing the active ingredient may be administered directly, e.g. by syringe or, more desirably, by infusion. For direct administration on aqueous solution containing up to 0.5 mg. per ml. of solution is convenient. For infusion the active ingredient is desirably diluted, for example, with isotonic glucose to one liter and administred gradually over a `6-8 hour period. This latter method eliminates or at least minimizes gastrointestinal symptoms which may occur on direct administration. Mithramycin C, on the other hand, is administered to tumor-bearing animals at a dosage level of from about 3 to about 20 mg./kg. of body weight. The liquid preparations, such as aqueous solutions, are particularly advantageous when the compound is employed in an amount of from about 0.1 to 2.5 mg./ml. of solution or suspension.

Preparations comprising pure mithramycin A or pure mithramycin B and mixtures thereof in any proportions can be used. The naturally occurring mixtures such as are produced by the herein described methods in which mithramycin A is the major active ingredient as well as artificially produced mixtures in which the proportions of mithramycins A and B may range from substantially pure A to substantially pure B are useful. From a practical standpoint, however, it is convenient to use the naturally occurring mixtures in which the proportions of mithramycin A to B and/or C range from about 85%-t 98% A to about l5%-2% B and/or C.

Mithramycin A is the drug of choice for the treatment of human patients having inoperable testicular tumors. In the case of one patient, an adult male, with an embryonal-type testicular tumor, complete remission occurred and the patient has been completely symptomfree for over 4 years.

Clinical investigators conducting earlier studies had recommended doses of 50 y/kg/day for 5 or more days. It was emphasized that the total dosage of mithramycin A had to be adjusted to the tolerance of each individual patient, that each patient had to be titrated in respect to the total dosage mithramycin A that the total dosage of mithramycin A could not be standardized as, for instance, in the case of nitrogen mustard, and that there is a wide range of sensitivity to the toxicity of mithramycin A. These doses were associated with formidable toxicity mainly in the form of an unexplained bleeding phenomenon which occurred in about 9% of treated patients. Subsequent studies revealed that with a 50% reduction in dosage (25 y/kg/day) the therapeutic results remained essentially the same as with the higher dosages while the incidence of morbidity, bleeding episodes, and mortality associated with mithramycin therapy was reduced to the point where treatment with mithramycin was acceptable and was associated with a minimum amount of toxicity in carefully selevted patients. Investigators using the currently recommended dosage regimen of mithramycin A are in agreement that the dose of 25 y/kg/ day is associated with a minimum of toxicity, an acceptable degree of morbidity, and a significantly decreased mortality.

The following examples are provided to illustrate specific processes for preparation of the mithramycin complex and separation thereof into the pure crystalline A, B, and C components. They are provided for illustra- 14 tive purposes and are not intended to limit the scope of the invention.

Example I A nutrient medium was prepared from the following materials in one liter of water:

Grams Glycerol l0 Corn starch l0 Hylrolyzed caseine (NZ amine B) 20 Distillers molasses solubles (Curbay B.G.) 5

The pH of the mixture was adjusted to 7 with potassium hydroxide and 5 grams of calcium carbonate was added followed by steam sterilization from about 30 to 40 minutes. A slant culture of the species S. argillaceus ATCC 12956 was transferred to 100 ml. of this medium in a 300 ml. Erlenmeyer flask and shaken 4-5 days until good growth was obtained. Inoculum for a larger fermentation was prepared by transferring the contents of the aforesaid flask under aseptic conditions to one liter of the same medium in a 3 liter ask and shaking the same for 48 hours.

Fifty gallons of nutrient medium having the composition indicated above were prepared, sterilized, and inoculated with the inoculum thus prepared. The organism was then cultivated under submerged conditions of aeration for a period of three days. The fermentation broth was filtered with the aid of Super Cel (diatomaceous earth) without adjustment of the pH. The lfiltrate was then adjusted to pH 2.0 with concentrated sulfuric acid and 10% sodium chloride was added per volume of filtrate.

The filtrate was then twice extracted with five gallons of methyl isobutyl ketone. The solvent extracts were separated, combined `and evaporated in vacuo to 500 ml. The resulting 500 ml. concentrate was next chromatographed on an acid-washed alumina column prepared with chloroform. The column was developed with chloroform, followed by a 95% chloroform-methanol system, and the major proportion of the mithramycin complex was removed with a chloroform-methanol system as a reddish-brown fraction.

Approximately 400 ml. of this fraction was evaporated in vacuo to 50 ml. and the resulting concentrate was again chromatographed on an acid-washed alumina column prepared with chloroform. The column was developed as above, i.e. 'with chloroform, 95% chloroform-methanol, and 90% chloroform-methanol, removing the mithramycin mixture which occurred as a bright yellow band. One-third of the resulting eluate was evaporated in vacuo to dryness, taken into Water, filtered, and, on evaporating to dryness, yielded 223 mg. of microcrystalline mithramycin complex. The remaining 2/3 was evaporated to dryness, taken into water, filtered and freeze-dried, yielding further product in the form of a bright yellow powder.

Example II A culture of S. plicatus ATCC No. 12957, was cultivated in a manner similar to that indicated in Example I, first in a 4liter glass pot and then in a 15G-gallon tank using a medium of the following composition:

Grams/liter Glucose (Cerelose) l0 Soy bean meal 15 Distillers solubles 2.5 Sodium chloride 2 Dipotassium phosphate 5 Calcium carbonate 2 Water to one liter.

The medium was adjusted to a pH of 7, sterilized, and inoculated with 5% by volume of a 48-hour culture grown as aforesaid. The fermentation was continued for a period of 45 hours.

Upon completion of the fermentation, the broth was filtered with about 5% Super Cel (diatomaceous earth 5 g,/ 100 cc. broth) and the cake was washed with 1/0 volume of Water. A sample of the filtrate was found to exhibit a high order of antimicrobialand tumor-inhibiting activity. The filtrate was adjusted to pH 6.0 and extracted once with 1/2 volume of n-butanol. The extract was concentrated in vacuo, with the addition of water to lower the distillation temperature, until it reached 1/2 of the original volume. Further concentration, without the addition of water, was carried out until -,O of the original volume was reached. The resulting concentrate was permitted to stand at 5 C. for 20 hours, during which time it deposited a tan-colored microcrystalline solid, herein designated as Fraction I. This fraction was separated by ltration and washed with butanol.

The butanol filtrate resulting from the above extraction was then concentrated further in the presence of water to remove most of the solvent. The dark colored residue which resulted was treated with 3% acetic acid and extracted three times with equal volumes of ethyl acetate. It was found that all except about 5 to 10% of the total microbiological activity passed into the solvent layer. The extract was concentrated to a small volume and added to a column of acid-washed alumina in ethyl acetate. Approximately 15 to 20 grams of alumina were employed per gram of crude solids in the extract, the solids content being about 5 to 10% of the extract.

The initial fractions from the column showed no antibacterial activity but on concentration yielded a colorless crystalline solid herein designated as Fraction IIv Thereafter, the mixture of mithramycins was eluted from the column with ethyl acetate, followed by 10% ethanol in ethyl acetate. The active fractions were combined and freed from solvent under reduced temperature, whereby the mithramycin mixture was obtained as a bright amorphous powder.

l 6 Example III Another fermentation was conducted, as in Example II, with a medium having the following composition:

Grams/ liter Glucose (Cerelose) 10 Soy bean meal l5 K2HPO4 5 NaCl 2 Distillers solubles 2.5

CaCOg 2 Water to one liter.

After adjusting to pH 7.0 and sterilizing, the medium was inoculated with the variant of S. plicatus described in Table III. The filtered broth was found to be active against Sarcoma 180 in mice, when tested as hereinafter described.

The mixture of mithramycins and the fractions that are produced therewith are quite distinct from one another, as evidenced by a number of their physical and chemical properties. Some of the more important of these properties are summarized in the following table.

TABLE XIV [Physical properties of mitliramycin mixture and fractions I and II] Fraction III, Fraction I Fraction II mithramyeiu mixture Property:

Color White White Lemon yellow. Crystal Structure Hexagonal plates Needles Microscopic prisms.

crnrl cm.-1 cin.-l

Ultraviolet Absorption solution).

(methanol TABLE XIV-Continued [ezln lello25 [alo2li Optical activity (methanol solution. 54.9".

gl:gg.(djni'ethylformamide. None Nom` Melting point 255-60" C. decomp 300 C l70-75 C. decomp.

g Oxygen (by difference) pK value (in water) Color with ferrie chloride None gielartln with diazonium chloride Negative Bright red Bright red.

o u "ty:

Slightly soluble in- Water lower alcohols, Most common organic Diethylether, benzene,

acetone. solvents, water. chloroform, amylaoetate, ethylene dlchloride, trichlorethylene. Soluble ln Hot water and mix- Pyridlne, dimethyl Water, lower alcohols.,

tures of water and formamide aqueous i.e. methyl, ethyl lower alcohols, pyri- N aOH, propyl, n-butyl, sec.

dine, dimethyl butyl, amyl, hexyl formamide aqueous alcohol, acetone,

NaOH. ethyl acetate,

methyl isobutyl ketone, benzyl alcohol.

Insoluble in Non-polar organic N ori-polar organic Carbon tetrachloride,

solvents, i.e. benzsolvents i.e. earbonpetroleum ether, ene carbon tetratetrachloride, benzcyclohexane.

chloride, petroleum ether. ether.

ene, petroleum Useful salts of purified mithramycin A and mithramycin B and mixtures thereof can be prepared by methods well known in the art, as by treatment of the products with an appropriate base, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, or the like, in aqueous solution or under anhydrous conditions. For example, the sodium salt can be prepared by dissolving the base in water and titrating to about pH 10 with sodium hydroxide and freeze drying the resulting solution. Other relatively strong bases may, of course, be employed to prepare the corresponding salts.

Example V An S. plcatus fermentation is conducted as described in AExample II, the whole broth adjusted to pH 4.0, and the mycelium filtered. An activated magnesium silicate (-60 mesh) column (Florisil, Floridin Co.) is prepared containing 75 g. of absorbent per gallon of fermentation broth. The ltered broth is than passed through the column at a feed rate of 30-40 gal. per hour. After the entire batch of filtered broth has been fed to the column, it is followed by water which washes the broth in the column through. The water wash is discontinued when the effluent is colorless or when the total solids contained therein falls to about l mg./ml. The solids content of effluent broth is about 2O mg./ml.

The column prepared in this fashion is then eluted with methanol. Fractions of convenient size are collected and their optical density at 280 mp. is measured. Those fractions having an optical density in excess of 2.0 are combined. A plate assay for activity against Staphylococcus aureus on each of these fractions is conducted.

The combined methanol eluate fractions containing active material are then concentrated to dryness, dissolved in a solvent mixture made up of three parts by volume of ethyl acetate and one part by volume of benzene. A chromatographic column having a 21/2 inch diameter and l5 inch length is then prepared employing a 2:1 mixture of silicio acid and cellulose powder (weight basis) as packing using the same solvent mixture in packing the column.

A portion of the ethyl acetate concentrate containing 5 to 8 g. of a mixture of mithramycins is then added to the column and the column developed with ethyl acetate collecting the eluate in 400 ml. fractions. Mithramycin A is rapidly eluted, traveling almost with the solvent front. The bulk of it is obtained in the iirst l() fractions (4 1.), but an additional 15 fractions (6 1.), containing a small amount of additional material due to tailing, are collected.

containing 5% by volume of methanol. Mithramycin B is collected in fractions 60--100. The progress of column development is readily followed visually either in ordinary development is readily followed visually either in ordinarv room light or ultra-violet light.

The fractions containing mithramycin A and mithramycin B are separately evaporated; the residues crystallized from n-butyl acetate or amyl acetate yielding the products in pure crystalline form. They exhibit the properties tabulated in Table lV.

Alternatively, the pure crystalline sodium salts of mithramycin A and mithramycin B can be prepared by concentration of the combined eluate fractions to a small volume and extraction thereof with water, adjustment of the water extracts to pH 8.0 with sodium hydroxide, freezing of the alkaline extract, and drying thereof from the frozen state. Other metal and organic base salts can be prepared in analogous fashion by substitution of the appropriate base. For instance, the calcium, magnesium, zinc, aluminum, ammonium, -hydroxyethylammonium, procaine, triethylammonium, ethylene diamine, potassium, lithium, ferric, and ferrous salts are thus prepared. The physical properties of the sodium salt of mithramycin A are listed in Table III.

Example VI A mixture of mithramycins A and B can be conveniently prepared in purified microcrystalline form from the methanolic eluate prepared as described in the first two paragraphs of Example V by concentration thereof to a low volume resulting in removal from the bulk of the methanol from the solution and extraction of the concentrate with ethyl acetate at pH 4.0. The ethyl acetate extracts are then combined and concentrated to a convenient volume for countercurrent distribution in a 100 tube automatic apparatus. They are distributed against 3% phosphate pH 7.0 to 7.5 buffer. The contents of the tubes are combined in groups of ten and assayed by the B. subtilis plate assay.

The bulk of the activity is found in tubes through 160. These fractions are combined and concentrated to a small volume, ether is added thereto and the mixture is extracted with water. The water layer is freed of solvent by evaporation, frozen, and dried from the frozen state. A yellow amorphous solid comprised of a mixture of approximately to 95% mithramycin A and 5 to 15% of mithramycin iB is obtained.

The pure crystalline sodium 'salt of mithramycin A is prepared from the aqueous extract described in the previous paragraph by adjusting it to pH 8.0 with sodium hydroxide and drying from the frozen state. The residue is crystallized from methanol or ethanol. A crystalline product is collected and recrystallized from 1:1 aqueous methanol yielding the pure crystalline dehydrated sodium salt as bright yellow elongated rectangular prismatic crystals.

Y Occasionally, during the course of countercurrent distribution in this fashion the sodium salts of a mixture of the mithramycins A and B separate in the tubes containing the highest concentration thereof. The sodium salts are moderately soluble in water but only slightly soluble in aqueous sodium chloride or sodium phosphate solutions. Their solubility in lower alkanols is very low and they are almost insoluble in the common organic solvents. The physical properties of pure crystalline sodium mithramycin A are listed in Table IV.

Example VII An S. piicatzls fermentation is conducted as described in Example II, the whole broth adjusted to pH 5 and the mycelium filtered. The filtrate is extracted with one-third volume n-butanol and the extract concentrated azeotropically. The concentrate is shaken between water and isopropyl ether (1:1), the phases separated and the aqueous phase brought to pH 7 with dilute sodium hydroxide solution. The neutralized solution is then freeze-dried and the residue extracted with ethanol. The ethanol extract is concentrated in vacuo to a thick solution whereupon the sodium salt of mithramycin A crystallizes and is filtered off. The ltrate is concentrated, transferred to ethyl acetate from an acid solution (pH 2-3) and adsorbed on a silicic acid column. Elution of the column with 5% methanol-ethyl acetate gives a mixture containing predorninantly mithramycin A with up to 5% of mithramycin B.

A mixture richer in mithramycin B is obtained by eluting the column with 25% methanol-ethyl acetate.

Example VIII The organism, Straptomyces plcatus ATCC 12957,

was grown in submerged culture in a medium composed of the following ingredients in grams per liter of water:

Glucose l Soy bean meal 15 Corn starch l0 NZ amine YTT Butyl molasses solubles 5 Sodium chloride 5 Calcium carbonate 2 A two to three day old vegetative inoculum was used to the extent of one to five percent and the fermentation carried out for about four days at 28-30 C. (The progress of the fermentation can be followed by a Bacillus subtilis plate assay at a suitable dilution.)

The culture liquid was filtered with the aid of one to two percent Hyflo Supercel at the existing pH (7.0-7.5). The filtrate was adjusted to pH 5.0-5.5 and extracted once with n-butanol (25 percent of the volume of the broth). The extract was separated, clarified and concentrated in the presence of water until almost all the solvent was removed. The concentrate was then shaken with an equal volume of isopropyl ether to remove oily impurities, then extracted twice with ethyl acetate at pH 2.0-3.0. The solvent extract was concentrated to a small volume and chromatographed on a column of acid-washed alumina (20 to 30 grams per gram of solids) made up in ethyl acetate. The sample was applied to the column and the latter eluted first with ethyl acetate and then with ethyl acetate containing increasing amounts of methanol. The bulk of the activity was recovered in the l0 to 20 percent methanol-ethyl acetate eluate. The compound was recovered by concentration of the active fractions and crystallized twice from acetone. Mithramycin A separates as a bright yellow crystalline solid.

The homogeneity of the product was demonstrated in the following thin layer chromatography system: Methyl ethyl ketone-isopropanol-methanol (8:1:1) Silica Gel G. Rf=0.60.

Further elution of the column with 25% methanolethylacetate affords predominantly mithramycin C and minor amounts of mithramycin B. This product is purified by chromatography on a silicio acid column (made up in ethyl acetate) followed by elution first with ethyl acetate and then with ethyl acetate containing increasing amounts of methanol. The C component is recovered from the 5 to 10% methanol-ethyl acetate eluate.

Example IX The fermentation procedure of Example VIII is followed and the products recovered by the following modified process.

The whole broth (approximately 1000 gallons) is filtered on an Oliver rotary filter, the pre-coat of which is neutralized with caustic soda to pH 6.0-7.0.

Fifty to one hundred pounds of lHyflo Supercel are added to the broth with agitation before filtration on the Oliver.

The filtrate is received in a tank suitable for solvent extraction. After filtration is complete the pH of the broth is adjusted to 5.0-5.5 with acid and saturated with nbutanol. The saturated broth filtrate is passed over a Podbielniack separator against n-butanol at 3 to l ratio.

The spent liquor (raffinate) is collected in a suitable tank and assayed against B subtilis. The raffinate is discarded if the assay indicates that the activity is low 20% of the original).

The butanol extract is sparkled, then concentrated in vacuo at 30-35 C. to remove the solvent, water being added throughout to aid in removal of the butanol. A solution of 0.1% dibasic phosphate buffer (Na+ or K+) is added to the vacuum pan to neutralize the residual acids and concentration continued until all the solvent is removed and the volume is less than 10% of the original volume.

The concentrate is then washed with isopropyl ether to remove oils present, re-concentrated to remove the isopropyl ether, then stirred with 10-15 sodium chloride. The pH is adjusted to 2-3 with acid, and the acidified concentrate extracted twice with ethyl acetate. The spent liquor (ethyl acetate raffinate) is then stirred with 5 lbs./ 50 gals. Hyflo Supercel and filtered.

The filter cake is suspended twice in methanol, filtration being carried out after each suspension. The spent liquor (after filtration) is assayed, and then discarded if potency is low.

The ethyl acetate-methanol filtrates are concentrated in vacuo (30-35 C.) then put on a Florisil column made up in ethyl acetate (l5-30 gms. per gm. solid). The column is eluted with ethyl acetate, then with ethyl acetate containing increasing amounts of methanol.

Most of the activity comes off in the 10-20% methanol fraction. The mithramycin A is then recovered by concentration of the active fractions, and crystallation of the solid obtained.

Activity in the broth, solvent extracts and column frac. tions can be followed by biological assay with Bacillus subtilis or Streptomyces aureus, or by optical density at 280 Ill/.4..

The B and C components are isolated by the procedure of Example VIII.

EXAMPLE X Purification and characterization of mithramycin C.- The fermentation procedure is the same as that described in Example II. The recovery procedure used is shown in the fiow sheet.

Filtered broth (150 gallons) Extract with 'Z5-35% u-butanol at pH 4-6 Cake Extract with two 3- liter portions of 25-50% methanol-ethyl acetate and ilter Filtrate Extract once with 5 liters ethyl acetate Cake- Filtrate (discard) Extract Combine and concentrate to about 2 4 liters Rainate (discard) The concentrate from the above flow sheet is stirred with 500-800 g. of Florisil and the slurry is diluted with 2-4 liters of ethyl acetate. The slurry is added to a column of Florisil (3-5 kg.) in ethyl acetate. The column is eluted successively with ethyl acetate, 20% and 40% methanol in ethyl acetate. The iirst two solvent eluates contain inert substances as shown by lack of activity against B. subtilis. The methanol-ethyl acetate fraction contains almost essentially mithramycin A fraction. The last eluate contains the residual quantities of mithramycin A and the bulk of mithramycin C.

Concentration of the 20% methanol-ethyl acetate fraction to a small volume (1.5-2.5 liters) yields a crystalline solid, the magnesium chelate of lmithramycin A. This can be recrystallized from methanol-ethyl acetate (1:3) for further purification.

The magnesium chelate crystallizes as large hexagonal prisms which decompose gradually at 210-215 C.

AllySlS.-Ca1cd fol' (C52H75024)2Mg.2`H20 (PCI'CCI): C, 56.04; H, 6.96; Mg, 1.09. Found (percent): C, 55.64; H, 7.19; Mg, 1.15.

When titrated in 50% aqueous methanol with hydrochloric acid, it shows a pKa value of 4.5-4.7 and an equivalent weight of 1110. The equivalent weight calculated fOl' (C52H75O24)2Mg. lS 14.

The crystalline magnesium chelate is converted to mithramycin A by stirring between water and ethyl acetate while adding hydrochloric acid to pH 3.0-4.0. The solvent layer is separated and the aqueous layer extracted twice more, preferably in the presence of 5-10% salt. The combined solvent layers are concentrated to dryness and the solid is crystallized from acetone. Mithramycin A separates as a bright yellow crystalline solid from acetone.

Titration in 50% aqueous methanol with sodium hydroxide shows a pKa value of 7.6i0.2 and an equivalent weight of 1115i15. (Calculated for C52H76O242H2O= 1 120) The mother liquors from the crystallization of the magnesium chelate are added to the 40% methanolethyl acetate eluate of the Florisil column and concentrated to a small volume. This concentrate is shaken with acid water (pH 3-4) to convert the sample to the free acid form. The Solvent layer is concentrated to remove most of the methanol and added to a column of silicic acid-cellulose (2:1) in ethyl acetate. About 15-25 g. of silicio acid is used per gram of the crude mithramycin as estimated by optical density at 280 ma using the relationship 50,000 optical density units equal 1 g. The column is eluted with ethyl acetate, 3-5% methanolethyl actate, 10% methanol-ethyl acetate. The progress of the column is followed by optical density and the solvents are changed as the need arises. Usually, mithramycin A appears with the ethyl acetate and, if the band moves too slowly, one can use the 3-5% methanolethyl acetate to speed its elution. The mithrarnycin C is eluted either with the 3-5% methanol-ethyl acetate or 10% methanol. The fractions are tested by thin layer chromatography in the system: methanolisopropanilmethyl ethyl ketone (111:8). Mithramycin A has an Rf of 0.6-0.7 while mithramycin C has an Rf of 0.15- 0.25. Fractions which contain mostly the C component are combined, concentrated to dryness and crystallized from acetone. Its solubility is similar to that of mithramycin A. Mithramycin B was not observed in this example.

The microbiological activity as well as other biological activities are of much lower degree than those of mithramycin A, as shown below. The values shown below are the diameters of the zones in the disc assay method using B. subtilis.

Salts of mithramycin C, such as the sodium, lithium, potassium and ammonium salts, are prepared according to the procedure of Example IV.

Example XI Several fermentations were carried out with S. plicatus (Parke-Davis & Company, Culture Bureau, Detroit, Mich., No. 04918) using the following medium.

Gms./liter Glucose (Cerelose) 10 Corn starch l0 Hydrolyzed casein (NZ Amine B 5 Distillers solubles 5 Sodium chloride 5 Soy bean meal 15 Calcium carbonate 1 Water to one liter.

The mixture was adjusted to pH 7 then sterilized. A plug from a slant culture of S. plctaus (Parke-Davis & Company Culture No. 04918) was placed in 200 ml. of this media in a shake liask and the ask shaken for three days. Two thousand mil. of this medium in a 4 l. pot was inoculated aseptically with 1% (20 ml.) of the inoculum from the shake flask. A series of such pots was set up and fermentation run for periods of 24, 48, 72 and 96 hours.

Upon completion of the fermentatin each of the broths was ltered and extracted with n-butanol at pH 5. The butanolic extractions were concentrated to near dryness. The concentrates were shaken with methanol, the methanolic solution iiltered then subjected to thin layer chromatography in the systems methyl ethyl ketone:isopro panol:methanol (8:1:l) silica gel G; and 0.02 N sodium chloride on diethylaminoethylcellulose.

The methanol filtrate was chromatographed on Florosil as described in Example V and the column eluted successively with methanol-ethyl acetate in which the methanol concentrations were 5, 10, 20 and 40%, respectively. The eluates were combined, concentrated to near dryness and the residue taken up in methanol. The ultraviolet spectrum of the eluates showed maximum absorption at approximately 262 ma. The mithramycins, as noted above, exhibit maximum absorption in the ultraviolet at 280 ma.

Thin layer chromatography of the concentrated eluates in the above mentioned systems, and paper chromatograms in the systems butanolzacetic acidzwater (5:1:4) and butanol saturated with water showed no mithramycins were present. All chromatograms were run against a mixture of mithramycins A, B and C for comparison.

The above experiments demonstrate that no mithramycin was produced by S. plcatus (Parke-Davis and Cornpany culture No. 04918).

What is claimed is:

1. A -method for the inhibition, in humans, of testicular tumors which comprises administering to said host an amount, effective for inhibiting said tumor, of mithramycin A, which is an acidic substance relatively soluble in water, lower alcohols, acetone, ethyl acetate and methyl isobutyl ketone, slightly soluble in diethyl ether and benzene, and substantially insoluble in petroleum ether, carbon tetrachloride and cyclohexane, which is capable of forming salts with bases, the pure crystalline dihydrate of which is yellow and contains the elements carbon, hydrogen, and oxygen in substantially the following percentages by weight:

Percent Carbon 55.5

Hydrogen 7.2 Oxygen 37.3

(By difference.)

and has the molecular formula C52H76O24-2H2O; which displays ultraviolet absorption maxima in methanol solution at 230 and 278 mit which has an equivalent weight of substantially 11151- and a pK,1 value of 7,610.2, which exhibits a dull yellow fluorescence when irradiated with ultraviolet light, and which as a 0.03% dispersion in potassium bromide exhibits absorption maxima in the infrared region of the spectrum at 736, 763, 803, 847, 901, 950, 978, 1000, 1064, 1118, 1160, 1228, 1259, 1293, 1320, 1370, 1396, 1440, 1500, 1580, 1626, 1720, 2850, 2910, 2950, 3390, cml, and which when dissolved in methanol at a concentration of 1% has the specific rotation -58; the crystalline anhydrous form of which is yellow melts at 184-187" C. and contains the elements carbon,` hydrogen and oxygen in substantially the following percentages by weight:

Percent Carbon 57.7 Hydrogen 7.2 Oxygen 35.1

(By difference.)

and has the molecular formula C52H76O24; and which displays ultraviolet absorption maxima in methanol solution at 230 and 278 my. and which as an 0.03% dispersion in potassium bromide exhibits absorption maxima in the infrared region of the spectrum at 806, 852, 908, 952, 983, 1004, 1071, 1126, 1170, 1236, 1266, 1300, 1330, 1374, 1515, 1631, 1724, 2890 and 3413 cml; and which on thin layer chromatography in the system methanolzisopropanolzmethyl ethyl ketone (1:128) has an Rf value of 0.6-0.7; the crystalline magnesium salt of which is bright yellow, decomposes at 210-215" C., has a pK,L value of 4.5-4.7 and an equivalent weight of 1110, and contains the elements carbon, hydrogen and magnesium in substantially the following percentages by weight:

Percent Carbon 55.64 Hydrogen 7.19 Magnesium 1.15

and has the molecular formula (C52H75O24)2Mg; the pure crystalline dihydrated sodium salt of which is bright yellow and contains the elements carbon and hydrogen in substantially the following percentages by weight:

Percent Carbon 54.65 Hydrogen 6.95

and has the molecular formula C52H75O24Na-2l-l20, which displays ultraviolet absorption maxima in methanol solution at 240, 285, 315, and 425 mp. and when dissolved in methanol at a concentration of 1% has the specific rotation +38.8.

2. The method of claim 1 wherein mithramycin A is administered at a dose equivalent to about 16 to 250 mcg/kg. of body weight per day.

3. The method of claim 1 wherein the tumor is embryonal testicular tumor.

4. The method of claim 3 wherein mithramycin A is administered at a dose equivalent to about 16 to 250 mcg./ kg. of body weight per day.

24 S. A method for the inhibition, in mice and rats, of tumors selected from the group consisting of:

human sarcoma No. 1

sarcoma mammary adenocarcinoma 755 human epidermoid carcinoma No. 3 lymphoid leukemia 1210 which comprises administering to said host an amount, effective for inhibiting said tumor, of a compound selected from the group consisting of mithramycin A, which is an acidic substance relatively soluble in water, lower alcohols, acetone, ethyl acetaten and methyl isobutyl ketone, slightly soluble in diethyl ether and benzene, and substantially insoluble in petroleum ether, carbon tetrachloride and cyclohexane, which is capable of forming salts with bases, the pure crystalline dihydrate of which is yellow and contains the elements carbon, hydrogen, and oxygen in substantially the following percentages by weight:

Percent Carbon 55.5 Hydrogen 7.2 Oxygen 37.3

(By difference.)

and has the molecular formula C52H76O242H2O; which displays ultraviolet absorption maxima in methanol solution at 230 and 278 mp. which has an equivalent weight of substantially 1115il5 and a pKE, value of 7.61 0.2, which exhibits a dull yellow uorescence when irradiated with ultraviolet light, and which as a 0.03% dispersion in potassium bromide exhibits absorpiton maxima in the infrared region of the spectrum at 736, 763, 803, 847, 901, 950, 978, 1000, 1064, 1118, 1160, 1228, 1259 1293, 1320, 1370, 1396, 1440, 1500, 1580, 1626, 1720, 2850, 2910, 2950, 3390, cin-1, and which when dissolved in. methanol at a concentration of 1% has the specic rotation -58; the crystalline anhydrous form of which is yellow melts at 184-187" C. and contains the elements carbon, hydrogen and oxygen in substantially the following percentages by weight:

Percent Carbon 57.7 Hydrogen 7.2 Oxygen 35.1

(By diierence.)

and has the molecular formula C52H76O24; and which displays ultraviolet absorption maxima in methanol solution at 230 and 278 mp. and which as an 0.03% dispersion in potassium bromide exhibits absorption maxima in the infrared region of the spectrum at 806, 852, 908, 952, 983, 1004, 1071, 1126, 1170, 1236, 1266, 1300, 1330, 1374, 1515, 1631, 1724, 2890 and 3413 cmrl; and which on thin layer chromatography in the system methanolzisopropanolzmethyl ethyl ketone (1 :118) has an Rf value of 0.6-0.7; the crystalline magnesium salt of which is bright yellow, decomposes at ZIO-215 C., has a pK,a value of 4.5-4.7 and an equivalent weight of 1110, and contains the elements carbon, hydrogen and magnesium in substantially the following percentages by weight:

Percent Carbon 55.64 Hydrogen 7.19 Magnesium 1.15

and has the molecular formula (C52H75O24)2Mg; the pure crystalline dihydrated sodium salt of which is bright yellow and contains the elements carbon and hydrogen in substantially the following percentages by weight:

Percent Carbon 54.65 Hydrogen 6.95

and has the molecular formula C52H75O24Na2H2O, which displays ultraviolet absorption maxima in methanol solution at 240, 285, 315, and 425 mit and when dissolved in methanol at a concentration of 1% has the specific rotation +38.8; mithramycin B, which is an acidic substance highly soluble in water, lower alcohols, acetone, ethyl acetate and methyl isobutyl ketone, slightly soluble in diethyl ether and benzene, and substantially insoluble in petroleum ether, carbon tetrachloride, and cyclohexane, which is capable of forming salts with bases, the pure crystalline form of which is orange-yellow and contains the elements carbon, hydrogen, and oxygen in substantially the following percentages by weight:

Percent Carbon 55.22 Hydrogen 7.13 Oxygen 37.65

(By difference.)

which displays ultraviolet absorption maxima is methanol solution at 230, 290, 320 and 410 ma, which exhibits a bright yellow iuorescence when irradiated with ultraviolet light, and which as a 0.03% dispersion in potassium bromide exhibits absorption maxima in the infrared region of the spectrum at 722, 743, 808, 847, 903, 948, 978, 1000, 1063, 1116, 1160, 1228, 1260, 1322, 1347, 1368, 1430, 1440, 1500, 1585, 1630, 1720, 2850, 2900, 2940, 3380 cm.1 and mithramycin C, an acidic substance soluble in water, lower alcohols, ethyl acetate, acetone and methyl isobutyl ketone, slightly soluble in diethyl ether and benzene, and substantially insoluble in petroleum ether, carbon tetrachloride and cyclohexane, which is capable of forming salts with bases, the pure crystalline form of which is bright yellow, melts at 182-184" C. and contains the elements carbon, hydrogen and oxygen in substantially the following percentages by weight:

Percent Carbon 55.25 Hydrogen 6.99 Oxygen 37.76

(By difference.)

which displays ultraviolet absorption maxima in methanol solution at 230 and 280 mp, and which as a 0.03% dispersion in potassium bromide exhibits absorption maxima in the infrared region of the spectrum at 848, 905, 980, 1020, 1070, 1166, 1233, 1266, 1299, 1408, 1515, 1634, 1706 and 3436 cm.1, and which on thin layer chromatography in the system methanol:isopropanolzmethyl ethyl ketone (l:1:8) has an Rf value of 0.150.25.

6. The method of claim 5 wherein mithramycin A or mithramycin B is administered yat ya dose equivalent to about 15 to about 250 mcg/kg. of body weight per day.

7. The method of claim 5 wherein mithramycin C is administered at a dose equivalent to about 3 to 20 mcg./ kg. body Weight per day.

References Cited Grundy et al.: Antibiotics and Chemotherapy, vol. III, No. 12 December 1953, pp. 1215-1216.

Philip et al.: Antibiotics and Chemotherapy, vol. III, No. l2 December 1953 pp. 1215-1216.

Berlin et al.: Nature, vol. 218, Apr. 13, 1969, pp. 193- 194.

Bakhaeva et al.: Tetrahedron letters No. 32, 1968, pp. 3595-3598.

Kennedy et al.: Cancer Chemotherapy Reports, No. 48, October 1965, pp. 59 to 63.

JEROME D. GOLDBERG, Primary Examiner T3550 UNITED STATES PATENT OFFICE `CER'THCA'FE 0F CORRECTN Patent No. 3,646,191* Dated February 29, 1972 Inventor(s) B. A., SObn et al It is certified that error appears in the above-identified patent and that said Letters Patent are nerebr corrected vas shown below:

Column ll, Table Il, in the column headed "BA-11028,"

line 25 opposite "Mannitel" change to i line 27 opposite "Rnamnose" change to line 28 opposite "Na acetate") change to line 29 opposite "Sorbitol") change to i Column 8, line 38, insert a colon after "7.5" Column 9, line l under Table III, change "C52H76O2".2H2.C," to

column 11, line 59, Change "celhorn" to Gelhorn Column 18, line 27 before "containing" insert The eluting solvent is then changed to ethyl acetate Column 18, delete line 30 as a duplication.

Column 2l, line 75, change isoproanil" to isopropanol Column 22, line im, change "mil," to ml,

Signed and sealed this 15th day of August 1972.

(SEAL) Attest:

EDWARD M. FLETCHER, JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents 

